The nicotinic alpha 4 receptor subunit contributes to the lining of the ion channel pore when expressed with the 5-HT3 receptor subunit

To understand the wide variation of calcium permeability seen in native and recombinant 5-HT3, receptor (5-HT3R) channels, we reported previously the novel hypothesis that the serotonin 5-HT3R subunit can co-assemble with the alpha 4 subunit of the nicotinic acetylcholine receptor (van Hooft, J. A., Spier, A. D., Yakel, J. L., Lummis, S. C. R. & Vijverberg, H. P. M. (1998) Proc. Natl. Acad, Sci. U. S. A. 95, 11456-11461), To test the hypothesis t hat the alpha 4 subunit contributes to the lining of the pore of the result ing 5-HT3R channel, a mutant nicotinic alpha 4 subunit with a reactive cyst eine residue engineered into the putative pore region was constructed by su bstituting the leucine at position 285 (alpha 4-L285C), The sulfhydryl-modi fying reagent [2-(trimethylammonium) ethyl]methanethiosulfonate (MTSET) red uced the acetylcholine-induced current in oocytes expressing this mutant ni cotinic (alpha 4-L285C subunit along with the nicotinic beta 2 subunit by s imilar to 60%. When the alpha 4-L285C subunit was co-expressed with the 5-H T3R subunit, both MTSET and silver nitrate (AgNO3), another cysteine-modify ing reagent, significantly reduced the serotonin-induced current, No reduct ion was seen when the 5-HT3R was expressed alone or with the wild-type alph a 4 subunit, These data provide direct molecular evidence that the nicotini c alpha 4 subunit co-assembles with the 5-HT3R subunit and forms an integra l part of the ion channel pore.